The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP3 receptor

Abstract

Although the presence of a BH4 domain distinguishes the antiapoptotic protein Bcl-2 from its proapoptotic relatives, little is known about its function. BH4 deletion converts Bcl-2 into a proapoptotic protein, whereas a TAT-BH4 fusion peptide inhibits apoptosis and improves survival in models of disease due to accelerated apoptosis. Thus, the BH4 domain has antiapoptotic activity independent of full-length Bcl-2. Here we report that the BH4 domain mediates interaction of Bcl-2 with the inositol 1,4,5-trisphosphate (IP3) receptor, an IP3-gated Ca(2+) channel on the endoplasmic reticulum (ER). BH4 peptide binds to the regulatory and coupling domain of the IP3 receptor and inhibits IP3-dependent channel opening, Ca(2+) release from the ER, and Ca(2+)-mediated apoptosis. A peptide inhibitor of Bcl-2-IP3 receptor interaction prevents these BH4-mediated effects. By inhibiting proapoptotic Ca(2+) signals at their point of origin, the Bcl-2 BH4 domain has the facility to block diverse pathways through which Ca(2+) induces apoptosis.

Fig. 1.

BH4 domain is necessary for Bcl-2-IP3 receptor interaction. (A) (Upper) Diagram depicting IP3 receptor type 1 and its functional domains. (Lower) Diagram depicting Bcl-2, its four BH domains and the C-terminal hydrophobic domain (TM). Diagrams are not drawn to scale. (B) GST pull down showing that BH4 deletion inhibits interaction of Bcl-2 with IP3 receptor domain 3 (d3). (Upper) Coomassie blue–stained gel showing GST-IP3 receptor fragment input levels. (Lower) Immunoblot showing input levels of Bcl-2 and ΔBH4Bcl-2 and Bcl-2 pulled down by IP3 receptor domain 3 (d3). (C) Co-immunoprecipitation of Bcl-2, but not ΔBH4Bcl-2, with the IP3 receptor. Type 1 IP3 receptor was immunoprecipitated from WEHI7.2 cells expressing either Bcl-2 or ΔBH4Bcl-2. (D) Binding of FITC-labeled TAT-BH4 (fTAT-BH4) to IP3 receptor domain 3. fTAT-BH4 (40 μM) was incubated with GST-IP3 receptor fragments corresponding to IP3 receptor domains 3 (GST-d3) and 6 (GST-d6) and the amount of fTAT-BH4 bound was quantified by fluorescence measurements. Negative controls included GST-EB (elution buffer), GST alone (GST), glutathione beads alone, GST-d3 alone, and FITC-TAT (fTAT). Symbols represent mean ± SEM of three experiments. (E) Co-immunoprecipitation of fTAT-BH4 peptide with IP3 receptor 1 in WEHI7.2 cell extracts. The WEHI7.2 cell extracts were incubated with 40 μM fTAT or fTAT-BH4 for 2 h before pull down by anti-fluorescein antibody. Immunoblot analysis was performed with anti-IP3 receptor 1 antibody.

Fig. 2.

BH4 peptide inhibits IP3 receptor channel activity. (A) BH4 peptide (without TAT) inhibits IP₃-induced Ca²⁺ release from the ER. A typical unidirectional ⁴⁵Ca²⁺-efflux experiment showing the Ca²⁺ release induced by 3 μM IP₃ from permeabilized ⁴⁵Ca²⁺-loaded wild-type MEF cells in the presence of vehicle (filled squares), 40 μM BH4 peptide (filled circles), 40 μM BH4 peptide, and 40 μM Pep2 (filled triangles) or 60 μM ctrl peptide (inverted filled triangles). All peptides were incubated from 4 min before the addition of IP₃ to 2 min after its addition (bars). Data points of a representative experiment, plotted as fractional loss (%/2 min) as a function of time, were obtained in duplicate and represent mean ± SD. Findings are representative of three independent experiments performed in duplicate. (B) Dose–response curve summarizing the effect of different concentrations of BH4 peptide and ctrl peptide on the IP₃-induced Ca²⁺ release from permeabilized wild-type MEF cells. Data points represent mean ± SEM, obtained from at least three independent experiments performed in duplicate and normalized to the amount of Ca²⁺ release provoked by IP3 under control conditions (vehicle). Logistic curve fitting indicates an IC₅₀ of about 32 μM for the BH4 peptide.

Fig. 3.

Inhibition of IP3-induced Ca²⁺ elevation by TAT-BH4 and reversal by TAT-Pep2. (A) Representative Ca²⁺ traces recording the Ca²⁺ elevation induced by 20 μg/ml anti-CD3 antibody in wild-type WEHI7.2 cells after pretreatment with 2 μM TAT-control (TAT-ctrl) or TAT-BH4 peptides for 1 h. Anti-CD3 was added 1–2 min after recording was started. (B) Histograms summarizing the average peak anti-CD3 induced Ca²⁺ elevation in WEHI7.2 cells treated with various peptides (mean ± SEM of seven individual experiments, >50 cells per sample per experiment). (C) Peak Ca²⁺ elevation induced by 20 μg/ml anti-CD3 antibody in wild-type Bcl-2(-) WEHI7.2 cells without any peptide addition, or in Bcl-2(+)WEHI7.2 cells in the absence of peptide and in the presence of either 10 μM TAT-ctrl or TAT-Pep2. Data are from three separate experiments (>50 cells per sample per experiment). Symbols represent mean ± SEM. (D) Peak Ca²⁺ elevation induced by 20 μg/ml anti-CD3 antibody in WEHI7.2 cells, either untreated or pretreated with 10 μM TAT-ctrl, 2 μM TAT-BH4, 2 μM TAT-BH4 + 10 μM TAT-Pep2, 2 μM TAT-BH4 + 10 μM TAT-ctrl (mean ± SEM of four separate experiments, >50 cells per sample per experiment). (E) Representative Ca²⁺ traces recording the Ca²⁺ elevation induced by 40 μM cell-permeable IP3 ester in wild-type Bcl-2 (-) WEHI7.2 cells after pretreatment with 1 μM TAT-ctrl or TAT-BH4 peptide for 1 h. IP3 ester was added 1–2 min after the recording was started. (F) Summary of the peak Ca²⁺ elevation induced by 50 μM IP3 ester in TAT-ctrl-and TAT-BH4-pretreated cells (mean ± SEM in three separate experiments, >50 cells per sample per experiment).

Fig. 4.

Inhibition of anti-CD3-induced apoptosis by TAT-BH4 and reversal by TAT-Pep2. (A) Representative Ca²⁺ traces recording the Ca²⁺ elevation induced by 5 μg/ml anti-CD3 antibody in Jurkat cells in the presence of 2 μM TAT-ctrl or TAT-BH4 peptides. Anti-CD3 was added 1–2 min after recording was started. (B) Histograms summarize the average peak anti-CD3 induced Ca²⁺ elevation in Jurkat cells treated with various peptides (mean ± SEM of four- individual experiments, >50 cells per sample per experiment). (C) TAT-BH4 or TAT-ctrl peptides were added at 1-μM final concentration 1 h before and 15 h after adding 5 μg/ml anti-CD3 antibody. Symbols represent the percentage of Hoechst 33342–stained cells displaying morphology typical of apoptosis 24 h after anti-CD3 addition (mean ± SEM; >200 cells counted per coverslip). (D) TAT-Pep2 reverses TAT-BH4-mediated inhibition of anti-CD3–induced apoptosis. Jurkat cells were incubated with or without TAT-BH4, and apoptosis was induced by anti-CD3 antibody as described in (C). Also, some of the cell suspensions were co-treated with 10 μM TAT-Pep2 or TAT-ctrl, added 1 h before anti-CD3 antibody, as outlined in the accompanying diagram. Symbols represent the percentage of cells (mean ± SEM) with apoptotic nuclei in four experiments (>200 cells counted per coverslip).